Fig. 4: The RNA stability and location of circDhx32 are regulated by its m6A modification level. | Acta Pharmacologica Sinica

Fig. 4: The RNA stability and location of circDhx32 are regulated by its m6A modification level.

From: Circular RNA circDhx32 promotes cardiac inflammatory responses in mouse cardiac ischemia-reperfusion injury via binding to FOXO1 competed with AdipoR1

Fig. 4

a The protein expression level of ALKBH5 in I/R-treated mice. n = 9. **P < 0.01 vs. Sham. P values were determined via an unpaired t test. b Western blot analysis of ALKBH5 in H/R-induced NMVCs. n = 6. **P < 0.01 vs. Ctrl. P values were determined via an unpaired t test. c The level of circDhx32 in cardiomyocytes transfected with si-NC or si-ALKBH5 and then subjected to Act D treatment at the indicated time points. n = 7. **P < 0.01 vs. Act D+si-NC. P values were determined via an unpaired t test. d RIP was performed to assess relative circDhx32 enrichment with a YTHDF2 antibody. n = 3. *P < 0.05 vs. Ctrl. P values were determined via an unpaired t test. e The expression of circDhx32 treated with Act D upon YTHDF2 overexpression. n = 5. *P < 0.05 vs. Act D+oe-NC. P values were determined via an unpaired t test. f The expression of circDhx32 in cells treated with Act D upon YTHDF2 silencing. n = 4. **P < 0.01 vs. Act D+si-NC. P values were determined via an unpaired t test. g After cotransfection with si-YTHDF2 and si-ALKBH5 for 48 h, cardiomyocytes were treated with Act D, and circDhx32 expression was quantified via qRT‒PCR at the indicated time points. n = 6. **P < 0.01 vs. Act D+si-NC; #P < 0.05 vs. Act D+si-ALKBH5+si-NC. One-way ANOVA followed by Dunnett’s multiple comparisons test was performed to evaluate significant differences. h The binding of circDhx32 to YTHDC1 was verified by a RIP assay. n = 3. *P < 0.05 vs. Ctrl. P values were determined via an unpaired t test. i Cytoplasmic and nuclear fractionation assays revealed the localization of circDhx32 in H/R-induced cardiomyocytes upon YTHDC1 upregulation. n = 5. *P < 0.05 vs. H/R+oe-NC. P values were determined via an unpaired t test. j FISH revealed that circDhx32 was located mainly in the nucleus and that overexpression of YTHDC1 reversed this phenomenon. Cy3 and DAPI were used to stain circDhx32 and the nucleus, respectively. Scale bar = 20 µm. n = 4‒7. The data are expressed as the means ± SEMs.

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